The histone code hypothesis proposes that cell fate decisions are achieved through creation of stable epigenetic histone marks at gene loci. These marks can be localized to promoters and transcribed regions of genes or can extend many kilobases beyond these boundaries. Although it is well established that certain histone marks are associated with transcriptional activation and other histone marks are associated with transcriptional repression, the precise mechanisms by which histone marks activate or repress transcription is incompletely understood. Further, it is becoming increasingly apparent that both activating and repressive marks are formed at loci of developmentally regulated genes and it is thought that these dual marks ensure developmental plasticity. For example, the Ifng gene exhibits complex activating and repressive patterns of epigenetic modifications that cover a region spanning over 50 kb of upstream and downstream genomic DNA in cells that express or silence Ifng. Given the critical role epigenetic marks play in normal development, it is becoming increasingly apparent that epigenetic defects also contribute to disease processes, including autoimmunity. Our results also demonstrate that failure to properly establish this long-range histone code may contribute to the characteristic over-production of IFN-3 by proliferating T cells from mice that develop autoimmune diabetes. To investigate these questions, we plan a three-pronged approach. First, we will prepare and analyze functional properties of transgenic mice with a wild-type human bacterial artificial chromosome (BAC) containing the IFNG gene and approximately 100 kb of flanking upstream and downstream sequence and BAC transgenic mice with various large (20-40 kb) and small (1kb) deletions within the 200 kb BAC. Second, we will perform detailed structure-function and nuclear positioning analyses to identify genomic sequences critical for these essential processes. Third, we will use several approaches to manipulate the formation stable long-range epigenetic histone marks across the IFNG locus and evaluate alterations in transcription, chromosomal conformation and nuclear positioning of the IFNG locus. Together, these studies will provide direct links between the function of the genetic code and the epigenetic code. They will also identify defects in the epigenetic code that may contribute to autoimmune disease. PUBLIC HEALTH RELEVANCE: The histone code hypothesis proposes that cell fate decisions are achieved through creation of stable epigenetic histone marks at gene loci. In this proposal, we plan to elucidate mechanisms underlying formation of long range histone marks across the Ifng locus in developing effector Th1 and Th2 cells and functional consequences created by these marks. Increasing evidence suggests that imbalance in the histone code may contribute to disease onset or pathogenesis, including autoimmune diseases, and it may be possible to affect the course of disease by altering the epigenetic code through increasing levels of activating histone acetylation marks or decreasing levels of inhibitory histone methylation marks, either generally or at specific genomic loci.